Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .

中文翻译：

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通过自诱导条件优化温度和碳源来增强大肠杆菌 BL21 (DE3) 中细胞外双特异性抗 MUC1 纳米抗体的表达

大肠杆菌是最适合生产抗体和抗体片段的宿主之一。向培养基中分泌抗体片段可提高细胞培养中的产品纯度并降低下游成本。在本研究中，选择了含有双特异性抗 MUC1 纳米抗体编码基因的大肠杆菌菌株 BL21 (DE3)，并研究了双特异性抗 MUC1 纳米抗体表达的自诱导方法。由于用乳糖代替IPTG作为诱导剂，在最终产品中观察到较少的杂质和毒性。为了增加细胞内和细胞外纳米抗体的产生，最初，对温度和蛋白质表达持续时间等关键因素进行了实验。在 33°C 下 21 小时后产生了最高量的纳米抗体。不同碳源、甘油、葡萄糖、乳糖和甘氨酸作为培养基添加剂在最佳温度和时间也使用响应面方法进行了评估。甘油、葡萄糖、乳糖和甘氨酸的碳源优化浓度分别为 0.75% (w/v)、0.03% (w/v)、0.1% (w/v) 和 0.75% (w/v) ， 分别。最后，评估了在优化的自诱导条件下在 2 L 发酵罐中生产纳米抗体。结果表明，87.66 µg/mL 抗 MUC1 纳米抗体的总滴度为 12.23 µg/L，大约是 LB 培养基中产生的纳米抗体总滴度的 7 倍。甘油、葡萄糖、乳糖和甘氨酸分别为 1% (w/v) 和 0.75% (w/v)。最后，评估了在优化的自诱导条件下在 2 L 发酵罐中生产纳米抗体。结果表明，87.66 µg/mL 抗 MUC1 纳米抗体的总滴度为 12.23 µg/L，大约是 LB 培养基中产生的纳米抗体总滴度的 7 倍。甘油、葡萄糖、乳糖和甘氨酸分别为 1% (w/v) 和 0.75% (w/v)。最后，评估了在优化的自诱导条件下在 2 L 发酵罐中生产纳米抗体。结果表明，87.66 µg/mL 抗 MUC1 纳米抗体的总滴度为 12.23 µg/L，大约是 LB 培养基中产生的纳米抗体总滴度的 7 倍。